Anatomic Pathology / HELICOBACTER PYLORI

Helicobacter pylori To Stain or Not to Stain? S. Brooks Smith, MD, Anthony N. Snow, MD, Randall L. Perry, JD, and Shadi A. Qasem, MD Key Words: Helicobacter pylori; Chronic gastritis; Gastric biopsies; Intestinal metaplasia; Lymphoid follicles DOI: 10.1309/AJCP8DGTAVG7MBMT

We performed a retrospective study to investigate the usefulness of immunohistochemical stains for the diagnosis of Helicobacter pylori (HP). We reviewed 200 consecutive gastric biopsy specimens, as well as immunohistochemical stains for HP. Of the biopsy specimens, 32 were positive for HP by immunohistochemical staining; of those, HP was seen on H&E stains in 29 cases (91%). The number of highpower fields required to detect HP on H&E-stained slides ranged from 1 to 25 (mean, 5.75). Combined significant (2+ or 3+) acute and chronic inflammation had a specificity of 98% and a negative predictive value of 97%. Our results show that, in our institution, HP can be seen relatively easily with H&E staining in the majority of cases; however, a small number of cases with significant inflammation can be missed if stains are not used.

More than 25 years ago, Marshall and Warren1 characterized a curved, gram-negative, flagellated bacillus found in biopsy specimens from the antral mucosa, ultimately named Helicobacter pylori (HP). The ensuing years have produced roughly 30,000 reports ranging from HP’s association with lymphoma to its unusual fatty acid substitution in lipids and lipopolysaccharides to the usefulness of barrier-born pigs as animal models for the study of HP induced gastritis! Ridiculous? Perhaps, but this amount of literature also illustrates the shared fascination and intrigue of many people for these bacteria. Accurate diagnosis of HP involves the combined knowledge, effort, and improved technology of laboratories, endoscopists, and pathologists. Yet, a set of eyes and a microscope are still considered a convenient and practical way of making this diagnosis. Since the discovery of HP, pathologists have used many diagnostic techniques while searching for a more sensitive and specific detection method. Among these competing techniques are immunohistochemical studies and special stains such as Giemsa and Warthin-Starry. For years, differing arguments have been developed about the need for and usefulness of these ancillary techniques. Some laboratories preemptively stain all gastric biopsy specimens, while others favor a more “judicious” application of ancillary studies. We retrospectively analyzed the use of immunohistochemical staining at our institution and reviewed the literature to assess the usefulness of preemptive staining of gastric biopsy specimens for the identification of HP.

Material and Methods Specimen Selection This was a retrospective study of 200 consecutive gastric biopsy specimens obtained from our files using the key words © American Society for Clinical Pathology

Am J Clin Pathol 2012;137:733-738 733

DOI: 10.1309/AJCP8DGTAVG7MBMT

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Abstract

Smith et al / HELICOBACTER PYLORI

dry-field microscopy, and the 40× objective was the highest power available. A minimum of 20 high-power fields (HPFs) per biopsy specimen were examined for the presence of HP, and the number of HPFs needed to view HP on H&E stains and immunostains was assessed.

Histologic Evaluation H&E staining was initially performed on biopsy material that had been formalin fixed and paraffin embedded. Progressive H&E staining was performed using the Leica Jung Autostainer XL (Leica Microsystems, Buffalo Grove, IL). Paraffin was removed using xylene followed by ethyl alcohol dehydration. The sections were rehydrated with tap water and stained with Gill hematoxylin (Gill et al2) for 4 minutes. After another tap water wash, Scott tap water (Luna3) was applied for 40 seconds. After an ethyl alcohol rinse, the eosin-phloxine stain (Luna3) was applied for 35 seconds. The sections were then dehydrated in ethyl alcohol, cleared with xylene, and coverslipped using a Richard-Allan mounting medium (Richard-Allan Scientific, Kalamazoo, MI). We performed immunohistochemical stains for HP (B-0471, dilution 1:200; DAKO, Carpinteria, CA) for all biopsy specimens in which no immunohistochemical stain had been initially ordered (82 cases). The H&E-stained and accompanying immunohistochemically stained slides were independently evaluated by 2 pathologists (S.B.S. and S.A.Q.). We recorded the type and degree of inflammation on a 3-digit scale (1, mild; 2, moderate; 3, severe) ❚Table 1❚. We graded neutrophils within the biopsy specimens as follows: 1, rare neutrophils (difficult to find); 2, small clusters of neutrophils (